The soil microbial biomass survives as a largely dormant population for long periods without fresh substrates, depending for growth upon a rapid uptake of substrates when they become available. Currently, little investigation has been made into the mechanisms involved in the transition from dormancy to activity. We found that additions of trace amounts of different simple and complex substrates (glutamic acid, amino acids mixture, glucose, protein hydrolysates, carbohydrates, compost extract), even at very low application rates (5-μg C g−1 soil), caused an immediate and significant activation (measured as increased CO2-C evolved) of the soil microbial biomass. The different substrates caused different intensities of respiration response, which were related to the substrates’ composition, complexity, and degradability. The difference between the CO2-C evolved from the amended soil minus that evolved from a similarly incubated but non-amended soil ranged from 80 to 160% of the humified carbon C added as substrate, with most of the substrates causing a positive priming effect, in agreement with previous findings. The activation ended after 5–70 h, depending on the substrate, but the microbial biomass could be reactivated with further additions. It seems that the microbial biomass first responds to traces of substrate by increasing its metabolic activity in anticipation of a larger ‘food event’. Overall, these results indicate that soil micro-organisms have evolved metabolic and physiological strategies that allow them to survive and growth in the generally poor-substrate soil environment.